The search for ultra sensitive fluorescent bio-probes for analytical and biophysical applications is very active (Bruchez M. et al, 1998; van de Rijke et al, 2001). To date, the commonly used fluorescent bio-probes are organic dyes, such as rhodamine, fluorescein isothiocyanates (FITC), and cyanine dyes (Cy3, Cy5 and Cy7). More recently, semiconductor nanoparticles (quantum dots, QDs) have been employed as bio-probes (Gerion D et al, 2001; Dubertret B et al, 2002). These down-conversion fluorescent bio-probes emit one lower energy fluorescent photon after absorbing another higher energy UV or visible photon. The main problem of these probes in bio-applications is the autofluorescence (noise) from the analytes under UV and visible light. It decreases the signal-to-noise ratio, limiting the sensitivity.
The use of infrared-to-visible up-conversion phosphors as bio-probe, which is able to absorb and combine two or more near-infrared (NIR) photons with lower energy to produce a higher energy photon in the visible spectrum, is a promising approach to solve the problem of autofluorescence. This concept was first disclosed in U.S. Pat. No. 5,674,698. Compared with the current bio-probes including organic dyes, fluorescent proteins and quantum dots, the advantages include: improved signal-to-noise ratio due to absence of autofluorescence and reduction of light scattering, the non-invasive 980 nm NIR excitation falls within the “water window” (a gap in the absorption spectrum of tissue between chromophores (<800 nm) and water (>1200 nm)). In vivo imaging can be easily achieved as a function of the strong tissue penetration ability of the NIR. Photo-bleaching can be greatly reduced because of the resistance to photo-bleaching of these inorganic nanoparticles. Multiple labelling can also be achieved by the fluorescent nanoparticles at various optical wavelengths under the same 980 nm NIR excitation.
Currently, the fabrication of up-conversion fluorescent inorganic nanoparticles suitable as bio-probe remains as the key technological issue. As bio-probes, the targeted molecules (such as proteins, oligonucleotides and other biomolecules in cells or tissues) are in the range from several nanometers to tens of nanometers. An optimal universal bio-probe therefore should be small in size with narrow size distribution. It should yield high fluorescent efficiency and must be water re-dispersible (Dubertret B et al, 2002).
The most efficient infrared-to-visible up-conversion phosphors are Yb—Er or Yb—Tm co-doped fluorides such as NaYF4, BaYF5, NaLaF4, NaGdF4, YF3, LaF3, GdF3 and oxysulphides like Y2O2S (Basse G and Grabmaier B C, 1994), where fluorides and oxysulphide are the hosts, ytterbium (Yb) acts as the sensitizer and erbium (Er) or thulium (Tm) acts as the fluorescent centre. Under the 980 nm NIR excitation, they give off different colours of visible up-conversion fluorescence, depending on the different doping ions. Among them, rare earth doped hexagonal phase NaYF4 is one of the most efficient material for green and blue up-conversion. However, all these commercially available phosphors are in bulk form usually prepared by high-temperature solid-state reactions. Making these bulk phosphors into nanoparticles, which simultaneously satisfy the bio-probe criteria mentioned above, remains a big challenge. Several research groups have sought alternative approaches and synthesized the up-conversion fluorescent nanoparticles for bio-probes. 400 nm Yb—Er and Yb—Tm co-doped Y2O2S up-conversion fluorescent particles have been adopted for detection of nucleic acid (van de Rijke, F et al, 2001). The particles were prepared using the method disclosed in U.S. Pat. No. 6,039,894. Their synthesized particles were, however, too large for application as bio-probes; and the efficiency of Y2O2S was less than that of the hexagonal phase NaYF4 phosphors. Fabrication of smaller particles is being researched on (Corstjens P et al. 2005). Several other research groups focused on the synthesis of doped NaYF4 nanoparticles.
In WO 03/087259 the synthesis of 37 nm NaYF4:Yb,Er up-conversion nanoparticles, by the room-temperature reaction of rare earth-EDTA complex with sodium fluoride in aqueous solution has been disclosed (Yi G S et al, 2004). The synthesis and multicolour up-conversion emission of Yb—Er and Yb—Tm co-doped NaYF4 nanoparticles has been reported (Heer S, et al, 2004). The 15 nm nanoparticles were acquired by the reaction of rare earth N-(2-hydroxyethyl)ethylenediamine salt, sodium alkoxide of N-(2-hydroxyethyl)ethylenediamine and N-(2-hydroxyethyl)ethylenediamine fluoride at 200° C. under dry N2 atmosphere for 2 h. Recently, Wang et al, 2005, reported a liquid-solid-solution (LSS) method for the synthesis of NaYF4:Yb,Er up-conversion nanoparticles. However, all of the above efforts produced cubic-phase nanoparticles, with efficiency of at least one order of magnitude less than the desirable hexagonal phase. Although Zeng J H et al, 2005, reported the synthesis of hexagonal phase NaYF4:Yb,Er(Tm) nanoparticles, the bigger size of approximately 50 nm was not sufficiently small for them to be used as bio-probes for smaller molecules.
Yi G S and Chow G M, 2005, described the synthesis of LaF3:Yb,Er, LaF3:Yb,Ho and LaF3:Yb,Tm nanoparticles of size 5.4 nm having potential applications as bio-probes, by reacting LaCl3, YbCl3, ErCl3/HoCl3/TmCl3 and NaF at a temperature of 72° C. The particles could be dispersed in organic solutions and formed a transparent colloid. With the 980 nm NIR excitation, the nanoparticles yielded different fluorescent emissions in the visible range. However, their up-conversion fluorescent was not efficient.
Accordingly, there is a need in this field of technique of improved fluorescent nano-structured materials.